The Arabidopsis mTERF-repeat MDA1 protein plays a dual function in transcription and stabilization of specific chloroplast transcripts within the psbE and ndhH operons.

The mTERF gene family encodes for nucleic acid binding proteins that are predicted to regulate organellar gene expression in eukaryotes. Despite the implication of this gene family in plant development and response to abiotic stresses, a precise molecular function was assigned to only a handful number of its c. 30 members in plants. Using a reverse genetics approach in Arabidopsis thaliana and combining molecular and biochemical techniques, we revealed new functions for the chloroplast mTERF protein, MDA1. We demonstrated that MDA1 associates in vivo with components of the plastid-encoded RNA polymerase and transcriptional active chromosome complexes. MDA1 protein binds in vivo and in vitro with specificity to 27-bp DNA sequences near the 5'-end of psbE and ndhA chloroplast genes to stimulate their transcription, and additionally promotes the stabilization of the 5'-ends of processed psbE and ndhA messenger (m)RNAs. Finally, we provided evidence that MDA1 function in gene transcription likely coordinates RNA folding and the action of chloroplast RNA-binding proteins on mRNA stabilization. Our results provide examples for the unexpected implication of DNA binding proteins and gene transcription in the regulation of mRNA stability in chloroplasts, blurring the boundaries between DNA and RNA metabolism in this organelle.

Phloem-Triggered Virus-Induced Gene Silencing Using a Recombinant Polerovirus.

The phloem-limited poleroviruses infect Arabidopsis thaliana without causing noticeable disease symptoms. In order to facilitate visual infection identification, we developed virus-induced gene silencing (VIGS) vectors derived from Turnip yellows virus (TuYV). Short sequences from the host gene AtCHLI1 required for chlorophyll biosynthesis [42 nucleotides in sense or antisense orientation or as an inverted-repeat (IR), or an 81 nucleotide sense fragment] were inserted into the 3' non-coding region of the TuYV genome to screen for the most efficient and robust silencing vector. All recombinant viruses produced a clear vein chlorosis phenotype on infected Arabidopsis plants due to the expression inhibition of the AtCHLI1 gene. The introduction of a sense-oriented sequence into TuYV genome resulted in a virus exhibiting a more sustainable chlorosis than the virus containing an IR of the same length. This observation was correlated with a higher stability of the sense sequence insertion in the viral genome. In order to evaluate the impact of the TuYV silencing suppressor P0 in the VIGS mechanism a P0 knock-out mutation was introduced into the recombinant TuYV viruses. They induced a similar but milder vein clearing phenotype due to lower viral accumulation. This indicates that P0 does not hinder the performances of the TuYV silencing effect and confirms that in the viral infection context, P0 has no major impact on the production, propagation and action of the short distance silencing signal in phloem cells. Finally, we showed that TuYV can be used to strongly silence the phloem specific AtRTM1 gene. The TuYV-derived VIGS vectors therefore represent powerful tools to easily detect and monitor TuYV in infected plants and conduct functional analysis of phloem-restricted genes. Moreover this example indicates the potential of poleroviruses for use in functional genomic studies of agronomic plants.

Large gene overlaps and tRNA processing in the compact mitochondrial genome of the crustacean Armadillidium vulgare.

A faithful expression of the mitochondrial DNA is crucial for cell survival. Animal mitochondrial DNA (mtDNA) presents a highly compact gene organization. The typical 16.5 kbp animal mtDNA encodes 13 proteins, 2 rRNAs and 22 tRNAs. In the backyard pillbug Armadillidium vulgare, the rather small 13.9 kbp mtDNA encodes the same set of proteins and rRNAs as compared to animal kingdom mtDNA, but seems to harbor an incomplete set of tRNA genes. Here, we first confirm the expression of 13 tRNA genes in this mtDNA. Then we show the extensive repair of a truncated tRNA, the expression of tRNA involved in large gene overlaps and of tRNA genes partially or fully integrated within protein-coding genes in either direct or opposite orientation. Under selective pressure, overlaps between genes have been likely favored for strong genome size reduction. Our study underlines the existence of unknown biochemical mechanisms for the complete gene expression of A. vulgare mtDNA, and of co-evolutionary processes to keep overlapping genes functional in a compacted mitochondrial genome.

Arabidopsis MZT1 homologs GIP1 and GIP2 are essential for centromere architecture.

Centromeres play a pivotal role in maintaining genome integrity by facilitating the recruitment of kinetochore and sister-chromatid cohesion proteins, both required for correct chromosome segregation. Centromeres are epigenetically specified by the presence of the histone H3 variant (CENH3). In this study, we investigate the role of the highly conserved γ-tubulin complex protein 3-interacting proteins (GIPs) in Arabidopsis centromere regulation. We show that GIPs form a complex with CENH3 in cycling cells. GIP depletion in the gip1gip2 knockdown mutant leads to a decreased CENH3 level at centromeres, despite a higher level of Mis18BP1/KNL2 present at both centromeric and ectopic sites. We thus postulate that GIPs are required to ensure CENH3 deposition and/or maintenance at centromeres. In addition, the recruitment at the centromere of other proteins such as the CENP-C kinetochore component and the cohesin subunit SMC3 is impaired in gip1gip2. These defects in centromere architecture result in aneuploidy due to severely altered centromeric cohesion. Altogether, we ascribe a central function to GIPs for the proper recruitment and/or stabilization of centromeric proteins essential in the specification of the centromere identity, as well as for centromeric cohesion in somatic cells.

Differential effects of viral silencing suppressors on siRNA and miRNA loading support the existence of two distinct cellular pools of ARGONAUTE1.

Plant viruses encode RNA silencing suppressors (VSRs) to counteract the antiviral RNA silencing response. Based on in-vitro studies, several VSRs were proposed to suppress silencing through direct binding of short-interfering RNAs (siRNAs). Because their expression also frequently hinders endogenous miRNA-mediated regulation and stabilizes labile miRNA* strands, VSRs have been assumed to prevent both siRNA and miRNA loading into their common effector protein, AGO1, through sequestration of small RNA (sRNA) duplexes in vivo. These assumptions, however, have not been formally tested experimentally. Here, we present a systematic in planta analysis comparing the effects of four distinct VSRs in Arabidopsis. While all of the VSRs tested compromised loading of siRNAs into AGO1, only P19 was found to concurrently prevent miRNA loading, consistent with a VSR strategy primarily based on sRNA sequestration. By contrast, we provide multiple lines of evidence that the action of the other VSRs tested is unlikely to entail siRNA sequestration, indicating that in-vitro binding assays and in-vivo miRNA* stabilization are not reliable indicator of VSR action. The contrasted effects of VSRs on siRNA versus miRNA loading into AGO1 also imply the existence of two distinct pools of cellular AGO1 that are specifically loaded by each class of sRNAs. These findings have important implications for our current understanding of RNA silencing and of its suppression in plants.

DOLICHOL PHOSPHATE MANNOSE SYNTHASE1 mediates the biogenesis of isoprenyl-linked glycans and influences development, stress response, and ammonium hypersensitivity in Arabidopsis.

The most abundant posttranslational modification in nature is the attachment of preassembled high-mannose-type glycans, which determines the fate and localization of the modified protein and modulates the biological functions of glycosylphosphatidylinositol-anchored and N-glycosylated proteins. In eukaryotes, all mannose residues attached to glycoproteins from the luminal side of the endoplasmic reticulum (ER) derive from the polyprenyl monosaccharide carrier, dolichol P-mannose (Dol-P-Man), which is flipped across the ER membrane to the lumen. We show that in plants, Dol-P-Man is synthesized when Dol-P-Man synthase1 (DPMS1), the catalytic core, interacts with two binding proteins, DPMS2 and DPMS3, that may serve as membrane anchors for DPMS1 or provide catalytic assistance. This configuration is reminiscent of that observed in mammals but is distinct from the single DPMS protein catalyzing Dol-P-Man biosynthesis in bakers' yeast and protozoan parasites. Overexpression of DPMS1 in Arabidopsis thaliana results in disorganized stem morphology and vascular bundle arrangements, wrinkled seed coat, and constitutive ER stress response. Loss-of-function mutations and RNA interference-mediated reduction of DPMS1 expression in Arabidopsis also caused a wrinkled seed coat phenotype and most remarkably enhanced hypersensitivity to ammonium that was manifested by extensive chlorosis and a strong reduction of root growth. Collectively, these data reveal a previously unsuspected role of the prenyl-linked carrier pathway for plant development and physiology that may help integrate several aspects of candidate susceptibility genes to ammonium stress.

Arabidopsis histone methyltransferase SET DOMAIN GROUP8 mediates induction of the jasmonate/ethylene pathway genes in plant defense response to necrotrophic fungi.

As sessile organisms, plants have to endure a wide variety of biotic and abiotic stresses, and accordingly they have evolved intricate and rapidly inducible defense strategies associated with the activation of a battery of genes. Among other mechanisms, changes in chromatin structure are thought to provide a flexible, global, and stable means for the regulation of gene transcription. In support of this idea, we demonstrate here that the Arabidopsis (Arabidopsis thaliana) histone methyltransferase SET DOMAIN GROUP8 (SDG8) plays a crucial role in plant defense against fungal pathogens by regulating a subset of genes within the jasmonic acid (JA) and/or ethylene signaling pathway. We show that the loss-of-function mutant sdg8-1 displays reduced resistance to the necrotrophic fungal pathogens Alternaria brassicicola and Botrytis cinerea. While levels of JA, a primary phytohormone involved in plant defense, and camalexin, a major phytoalexin against fungal pathogens, remain unchanged or even above normal in sdg8-1, induction of several defense genes within the JA/ethylene signaling pathway is severely compromised in response to fungal infection or JA treatment in mutant plants. Both downstream genes and, remarkably, also upstream mitogen-activated protein kinase kinase genes MKK3 and MKK5 are misregulated in sdg8-1. Accordingly, chromatin immunoprecipitation analysis shows that sdg8-1 impairs dynamic changes of histone H3 lysine 36 methylation at defense marker genes as well as at MKK3 and MKK5, which normally occurs upon infection with fungal pathogens or methyl JA treatment in wild-type plants. Our data indicate that SDG8-mediated histone H3 lysine 36 methylation may serve as a memory of permissive transcription for a subset of defense genes, allowing rapid establishment of transcriptional induction.

An endogenous, systemic RNAi pathway in plants.

Recent work on metazoans has uncovered the existence of an endogenous RNA-silencing pathway that functionally recapitulates the effects of experimental RNA interference (RNAi) used for gene knockdown in organisms such as Caenorhabditis elegans and Drosophila. The endogenous short interfering (si)RNA involved in this pathway are processed by Dicer-like nucleases from genomic loci re-arranged to form extended inverted repeats (IRs) that produce perfect or near-perfect dsRNA molecules. Although such IR loci are commonly detected in plant genomes, their genetics, evolution and potential contribution to plant biology through endogenous silencing have remained largely unexplored. Through an exhaustive analysis performed using Arabidopsis, we provide here evidence that at least two such endogenous IRs are genetically virtually indistinguishable from the transgene constructs commonly used for RNAi in plants. We show how these loci can be useful probes of the cellular mechanism and fluidity of RNA-silencing pathways in plants, and provide evidence that they may arise and disappear on an ecotype scale, show highly cell-specific expression patterns and respond to various stresses. IR loci thus have the potential to act as molecular sensors of the local environments found within distinct ecological plant niches. We further show that the various siRNA size classes produced by at least one of these IR loci are functionally loaded into cognate effector proteins and mediate both post-transcriptional gene silencing and RNA-directed DNA methylation (RdDM) of endogenous as well as exogenous targets. Finally, and as previously reported during plant experimental RNAi, we provide evidence that endogenous IR-derived siRNAs of all size classes are not cell-autonomous and can be transported through graft junctions over long distances, in target tissues where they are functional, at least in mediating RdDM. Collectively, these results define the existence of a bona fide, endogenous and systemic RNAi pathway in plants that may have implications in adaptation, epiallelism and trans-generational memory.

Mouse cytomegalovirus microRNAs dominate the cellular small RNA profile during lytic infection and show features of posttranscriptional regulation.

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Originally identified in a variety of organisms ranging from plants to mammals, miRNAs have recently been identified in several viruses. Viral miRNAs may play a role in modulating both viral and host gene expression. Here, we report on the identification and characterization of 18 viral miRNAs from mouse fibroblasts lytically infected with the murine cytomegalovirus (MCMV). The MCMV miRNAs are expressed at early times of infection and are scattered in small clusters throughout the genome with up to four distinct miRNAs processed from a single transcript. No significant homologies to human CMV-encoded miRNAs were found. Remarkably, as soon as 24 h after infection, MCMV miRNAs constituted about 35% of the total miRNA pool, and at 72 h postinfection, this proportion was increased to more than 60%. However, despite the abundance of viral miRNAs during the early phase of infection, the expression of some MCMV miRNAs appeared to be regulated. Hence, for three miRNAs we observed polyuridylation of their 3' end, coupled to subsequent degradation. Individual knockout mutants of two of the most abundant MCMV miRNAs, miR-m01-4 and miR-M44-1, or a double knockout mutant of miR-m21-1 and miR-M23-2, incurred no or only a very mild growth deficit in murine embryonic fibroblasts in vitro.

Intra- and intercellular RNA interference in Arabidopsis thaliana requires components of the microRNA and heterochromatic silencing pathways.

In RNA interference (RNAi), double-stranded RNA (dsRNA) is processed into short interfering RNA (siRNA) to mediate sequence-specific gene knockdown. The genetics of plant RNAi is not understood, nor are the bases for its spreading between cells. Here, we unravel the requirements for biogenesis and action of siRNAs directing RNAi in Arabidopsis thaliana and show how alternative routes redundantly mediate this process under extreme dsRNA dosages. We found that SMD1 and SMD2, required for intercellular but not intracellular RNAi, are allelic to RDR2 and NRPD1a, respectively, previously implicated in siRNA-directed heterochromatin formation through the action of DCL3 and AGO4. However, neither DCL3 nor AGO4 is required for non-cell autonomous RNAi, uncovering a new pathway for RNAi spreading or detection in recipient cells. Finally, we show that the genetics of RNAi is distinct from that of antiviral silencing and propose that this experimental silencing pathway has a direct endogenous plant counterpart.